Biomedical subjects

Mary E Klotman

Publications and source records attributed to Mary E Klotman.

At least 19 recordsLinked to original sources

PubMed2006

Retroviral E-DNA: persistence and gene expression in nondividing immune cells.

Following retroviral infection of cells, not only is the proviral DNA integrated into the host genome, but there is also an accumulation of unintegrated extrachromosomal DNA (E-DNA), both linear and circular. Although the integrated DNA is responsible for the production of viral proteins and new viral progeny, the role of E-DNA has remained uncertain. Several reports have shown that E-DNA is transcriptionally active producing both RNA, as well as viral proteins and that circular E-DNA can persist in nondividing cells, raising questions regarding the potential consequences of this reservoir. Furthermore, integrase inhibitors, presently in clinical trials, shifts the balance of proviral DNA to the E-DNA form. This review is focused on recent work in this field with an emphasis on exploring the potential role of E-DNA in both pathogenesis of retroviral infections, especially HIV-1, and as a tool to deliver and express genes.

Animals↗

PubMed2006

ACIDFORM inactivates herpes simplex virus and prevents genital herpes in a mouse model: optimal candidate for microbicide combinations.

The acidic vaginal milieu is presumed to inactivate pathogens but is neutralized by semen. This notion fostered the development of acid-buffering products, such as ACIDFORM (developed by Program for Topical Prevention of Conception and Disease, Rush University, and licensed by Instead), as microbicides. However, the extent and mechanism of protective activity provided by buffering gels is not known. Exposure of herpes simplex virus (HSV) to pH 4.5 or lower irreversibly inactivated HSV and reduced HSV yields by at least 90%; exposure to pH 5.0 had little or no effect. Pretreatment of HSV-2 with pH 3.5-4.5 triggered proteolysis, disrupting the HSV particle and resulting in a reduction in binding and invasion. ACIDFORM protected 21 (81%) of 26 mice from genital herpes, compared with 3 (12%) of 25 mice who received a placebo gel. ACIDFORM retained significant activity if mice were challenged with HSV delivered in seminal fluid. These findings suggest that ACIDFORM offers considerable protection against HSV and may be an optimal candidate for developing combination microbicides.

Animals↗

PubMed2006

Gonococcal lipooligosaccharide suppresses HIV infection in human primary macrophages through induction of innate immunity.

Gonorrhea often occurs as a coinfection with human immunodeficiency virus (HIV). Lipooligosaccharide (LOS) is a component of the gonococcal outer membrane that induces innate immunity through engagement of Toll-like receptor 4 (TLR4). We investigated the effects that LOS from 5 different strains of Neisseria gonorrhoeae have on HIV infection and on HIV provirus in primary human macrophages. LOS-treated human primary macrophages developed resistance to new HIV infection as well as to HIV provirus. Gonococcal LOS from the 5 strains and lipopolysaccharide (LPS) from Escherichia coli showed no significant difference in their anti-HIV activities. Suppression of HIV provirus resulted from the induction of interferon (IFN)-beta and subsequent activation of signal transducer and activator of transcription 1. Neutralization of IFN-beta , but not IFN-alpha , via antibody significantly reduced the anti-HIV activity induced by LOS and LPS. We conclude that LOS expressed by various strains of N. gonorrhoeae induce specific innate immune responses through TLR4 signaling, resulting in anti-HIV activity in human primary macrophages in vitro.

Gonorrhea↗

PubMed2006

Acute renal failure in hospitalized patients with HIV: risk factors and impact on in-hospital mortality.

BACKGROUND: Kidney disease is an increasingly important complication of HIV. OBJECTIVES: To examine the incidence and predictors of acute renal failure before and after the introduction of HAART, and the impact of acute renal failure on in-hospital mortality in the post-HAART era. METHODS: Adults hospitalized in acute care hospitals in New York State during 1995 (pre-HAART) or 2003 (post-HAART) were identified from the state Planning and Research Cooperative System database. HIV status was defined by primary or secondary diagnosis code. The impact of HIV and HAART on the incidence of acute renal failure and mortality, and the impact of acute renal failure on mortality, was assessed using chi analysis and multivariate regression. RESULTS: There were 52,580 HIV-infected patients discharged from hospital in 1995 and 25,114 in 2003. Compared with uninfected patients, HIV-infected patients had an increased incidence of acute renal failure in both the pre-HAART [adjusted odds ratio (OR), 4.62; 95% confidence interval (CI), 4.30-4.95] and post-HAART eras (adjusted OR, 2.82; 95% CI, 2.66-2.99). In the post-HAART cohort, acute renal failure was associated with traditional predictors such as age, diabetes mellitus, and chronic kidney disease, as well as acute or chronic liver failure or hepatitis coinfection (P < 0.001 for all comparisons). Acute renal failure was associated with mortality among HIV-infected patients in the post-HAART era (OR, 5.83; 95% CI, 5.11-6.65). CONCLUSIONS: Acute renal failure remains common among hospitalized patients with HIV and is associated with chronic kidney disease, liver disease, and increased mortality.

Acute Kidney Injury↗

PubMed2006

Dual tropism of HIV-1 envelopes derived from renal tubular epithelial cells of patients with HIV-associated nephropathy.

The phenotype of HIV-1 gp120 envelope derived from renal epithelium and peripheral blood mononuclear cells (PBMC) of patients with HIV-associated nephropathy was investigated in vitro. Chimeric viruses were derived from kidney or blood and used to infect primary CD4+T cells, cell lines expressing single co-receptors and a renal epithelial cell line HPT-1. HIV-1 variants derived from renal epithelium were dual tropic whereas simultaneously derived viruses from PBMC were R5-tropic. Utilization of alternative co-receptors CCR3, BONZO and BOB, also differed.

AIDS-Associated Nephropathy↗

PubMed2006

Role of ubiquitin-like protein FAT10 in epithelial apoptosis in renal disease.

Dysregulated apoptosis of renal tubular epithelial cells (RTEC) is an important component of the pathogenesis of several renal diseases, including HIV-associated nephropathy (HIVAN), the most common cause of chronic kidney failure in HIV-infected patients. In HIVAN, RTEC become infected by HIV-1 in a focal distribution, and HIV-1 infection has been shown to induce apoptosis in vitro. In microarray studies that used a novel renal tubular epithelial cell line from a patient with HIVAN, it was found that the ubiquitin-like protein FAT10 is one of the most upregulated genes in HIV-infected cells. Previously, FAT10 was shown to induce apoptosis in murine fibroblasts. The expression of FAT10 in HIVAN and the ability of FAT10 to induce apoptosis in human RTEC therefore were studied. This study revealed that FAT10 expression is induced after infection of RTEC by HIV-1 and that expression of FAT10 induces apoptosis in RTEC in vitro. Moreover, it was found that inhibition of endogenous FAT10 expression abrogated HIV-induced apoptosis of RTEC. Immunohistochemical studies demonstrated increased FAT10 expression in a murine model of HIVAN, in HIVAN biopsy samples, and in autosomal dominant polycystic kidney disease, another renal disease that is characterized by cystic tubular enlargement and epithelial apoptosis. These results suggest a novel role for FAT10 in epithelial apoptosis, which is an important component of the pathogenesis of many renal diseases.

AIDS-Associated Nephropathy↗

PubMed2006

Defensins in innate antiviral immunity.

Defensins are small antimicrobial peptides that are produced by leukocytes and epithelial cells, and that have an important role in innate immunity. Recent advances in understanding the mechanisms of the antiviral action(s) of defensins indicate that they have a dual role in antiviral defence, acting directly on the virion and on the host cell. This Review focuses on the antiviral activities and mechanisms of action of mammalian defensins, and on the clinical relevance of these activities. Understanding the complex function of defensins in innate immunity against viral infection has implications for the prevention and treatment of viral disease.

Animals↗

PubMed2006

HIV-1 infection initiates an inflammatory cascade in human renal tubular epithelial cells.

HIV-associated nephropathy (HIVAN) is the most common cause of chronic renal failure in HIV-infected patients. Tubulointerstitial inflammation is a prominent component of the histopathology of HIVAN. The pathogenesis of HIVAN is a result of infection of renal epithelial cells, but the cellular response to this infection remains poorly defined. In these studies, we used oligonucleotide microarrays to identify differentially expressed genes in renal tubular epithelial cells from a patient with HIVAN at three time points after infection with vesicular stomatitis virus-pseudotyped gag/pol-deleted HIV-1. Very few genes were differentially expressed 12 and 24 hours after infection. Three days after infection, however, 47 genes were upregulated by at least 1.8-fold. The most prominent response of these cells to HIV-1 expression was production of proinflammatory mediators, including chemokines, cytokines, and adhesion molecules. Many of the upregulated genes are targets of interleukin 6 and nuclear factor kappa B regulation, suggesting a central role for these proteins in the response of tubular epithelial cells to HIV-1 infection. Analysis of kidneys from HIV-1 transgenic mice revealed upregulation of many of the proinflammatory genes identified in the microarray studies. These studies provide novel insights into the mechanisms by which HIV-1 infection of tubular epithelial cells leads to tubulointerstitial inflammation and progressive renal injury.

AIDS-Associated Nephropathy↗

PubMed2006

Topical microbicides: the time has come.

Microbicides are vaginally or rectally applied drugs that inhibit HIV and other sexually transmitted pathogens. About 30 microbicidal products are being studied in preclinical and clinical trials. Data from in vitro, animal, and in vivo investigations with candidate microbicidal compounds support a proof of concept, although large-scale clinical trials will determine efficacy. Because there are multiple targets for HIV infection in the genital tract, synergistic combinations of microbicides will probably offer the greatest protection for non-HIV-infected persons and might decrease transmissibility when used by those infected with HIV.

Administration, Topical↗

PubMed2005

HIV-1 Nef induces dedifferentiation of podocytes in vivo: a characteristic feature of HIVAN.

OBJECTIVE: To determine the specific role of Nef in the pathogenesis of HIV-associated nephropathy. DESIGN: Podocytes are highly differentiated non-dividing cells in the normal glomerulus, however, they undergo dedifferentiation and acquire a proliferative phenotype in HIVAN patients, in HIV-transgenic mice and if infected by HIV-1 in vitro. These changes are accompanied by loss of the maturation markers synaptopodin and WT1, and expression of the proliferation marker Ki-67. Previously, we mapped the gene responsible for these changes in vitro to HIV-1 Nef. To determine the role of Nef in vivo, we developed a transgenic mouse model in which Nef was exclusively expressed in podocytes. METHODS: Transgenic mice were generated using a construct in which Nef expression was blocked by a floxed lacZ intervening gene. When crossed with another transgenic mice expressing Cre under the Podocin promoter (a podocyte specific gene), the intervening lacZ gene was removed activating the expression of Nef in podocytes. The in vivo expression profiles of the Nef, the proliferation marker Ki-67, the differentiation markers synaptopodin and WT1, and phospho-Stat3, were determined by immunohistochemistry. RESULTS: Podocyte-specific expression of Nef induced loss of synaptopodin and WT1, and expression of Ki-67 in podocytes. Furthermore, Nef activated expression of phospho-Stat3, one of the downstream signaling pathways for cell proliferation. CONCLUSIONS: We conclude that Nef induces the early molecular changes in podocytes that are essential for the dedifferentiation and proliferation of podocytes in HIVAN pathogenesis. These data provide the first clear molecular evidence that Nef alters the podocyte phenotype in vivo.

Animals↗

PubMed2005

PRO 2000 gel inhibits HIV and herpes simplex virus infection following vaginal application: a double-blind placebo-controlled trial.

BACKGROUND: Microbicides used to prevent the transmission of human immunodeficiency virus (HIV) are advancing to clinical trials on the basis of activity observed in vitro and in animal models. However, no data demonstrate activity of microbicides after application in humans. This study was designed to determine the antiviral activity in cervicovaginal lavage (CVL) samples collected after intravaginal application of 0.5% PRO 2000 gel (Indevus). METHODS: A randomized, double-blind study was conducted to assess the anti-HIV and anti-herpes simplex virus (HSV) activity of PRO 2000 in CVL samples obtained at screening (48 hours before) and 1 hour after application of study or placebo gel. HeLa cells or human macrophages were inoculated with CVL samples spiked with replication-defective HIV containing a luciferase indicator gene and pseudotyped with an R5 envelope. Human cervical epithelial cells were inoculated with CVL samples and challenged with HSV-2(G), and the virus titer was then determined. RESULTS: CVL samples obtained after application of PRO 2000 gel significantly inhibited HIV and HSV infection by at least 1000-fold, compared with CVL samples obtained at screening (P < .001). There were no differences in cytokine levels between the drug and placebo groups. CONCLUSIONS: PRO 2000 gel (0.5%) is sufficiently bioavailable and retains substantial antiviral activity after intravaginal application. This strategy provides a mechanism for testing the efficacy of a microbicide before embarking on large-scale clinical trials.

Administration, Intravaginal↗

PubMed2005

Cervicovaginal secretions contribute to innate resistance to herpes simplex virus infection.

Defining and preserving the innate antiviral activity found in cervicovaginal secretions is critical. Cervicovaginal lavage (CVL) samples were obtained from 20 healthy women and evaluated for anti-herpes simplex virus (HSV) activity. CVL samples reduced HSV-2 yields by 23-fold (median), and the anti-HSV activity of CVL samples correlated with the concentration of human neutrophil peptides (HNP)-1-3. Both CVL samples and HNP-1-3 interacted with virus and prevented entry after binding. Substantially less protective activity was observed in CVL samples obtained from 20 human immunodeficiency virus--infected subjects, but the addition of CVL samples from healthy subjects enhanced the antiviral activity. The significance of the innate activity was further demonstrated by showing that CVL samples prevented murine genital herpes. Fourteen of 15 mice were protected from genital herpes if they were challenged with HSV-2 pretreated with CVL samples from healthy subjects. In contrast, all 15 mice challenged with untreated HSV-2 died. These findings are evidence that cervicovaginal secretions contribute to innate resistance to HSV-2 and identify defensins as contributors to this activity.

Adult↗

PubMed2005

Highly active antiretroviral therapy and the epidemic of HIV+ end-stage renal disease.

The rise in the number of patients with HIV-associated nephropathy and HIV-infection with end-stage renal disease (HIV+ ESRD) continues to be a substantial concern for the ESRD program. In order to assess the impact of highly active antiretroviral therapy (HAART) on the progression of patients with AIDS to the development of ESRD and to project the prevalence of HIV+ ESRD through 2020, a mathematical model of the dynamics of HIV+ infection in the ESRD population was developed. Epidemiologic data on AIDS and HIV+ ESRD among black individuals in the United States were obtained since 1991 from the Centers for Disease Control and Prevention and US Renal Data System, respectively. The model was constructed to predict the prevalence of HIV+ ESRD incorporating the current rate of growth in AIDS prevalence. Two possible trends were considered: linear AIDS growth and exponential AIDS growth. The likely effectiveness of HAART in slowing progression to HIV+ ESRD was estimated from the best fit of the model to the data after 1995, when HAART was introduced. The model was then used to evaluate recent data and to project the prevalence of HIV+ ESRD through 2020. The model suggested that HAART has reduced the rate of progression from AIDS to HIV+ ESRD by 38%. The model projected an increase in HIV+ ESRD prevalence in the future as a result of the increase in the AIDS population among black individuals. This increase was predicted even assuming a 95% reduction in the progression from AIDS to HIV+ ESRD. Despite the potential benefit of HAART, the prevalence of HIV+ ESRD in the United States is expected to rise in the future as a result of the expansion of the AIDS population among black individuals. It is concluded that prevention of progression to ESRD should focus on early antiretroviral treatment of HIV-infected patients who have evidence of HIV-associated nephropathy.

AIDS-Associated Nephropathy↗

PubMed2005

HIV-1 extrachromosomal 2-LTR circular DNA is long-lived in human macrophages.

HIV-1 extrachromosomal 2-LTR circles (cc2LTR) are rapidly lost in dividing cell populations and, therefore, might be interpreted as representing new infection and ongoing viral replication. However, recent work demonstrated that cc2LTR persist in infected, growth-arrested T cell lines beyond their predicted half-life as previously determined in dividing cell populations. In this study, the evaluation of the stability of cc2LTR was extended to include primary human macrophages, a natural, non-dividing target of HIV-1. By quantitative real-time PCR, cc2LTR were found to persist out to 21 days post-infection in macrophages infected with both integrase competent and integrase- defective, recombinant HIV-1, whereas in activated CD4(+) T lymphocytes, they rapidly decreased over time. This persistence was associated with persistent, low level expression of the indicator gene, luciferase. These data suggest that the presence of HIV-1 cc2LTR in the PBMC of HIV-1-infected patients on suppressive HAART could be due either to ongoing generation of newly infected dividing cells, or persistence of circles in non-dividing cell populations where they appear to be stable. Furthermore, exrachromosomal circular DNA in this cell population could be a source of persisent viral protein expression.

Cells, Cultured↗

PubMed2005

Nef expressed from human immunodeficiency virus type 1 extrachromosomal DNA downregulates CD4 on primary CD4+ T lymphocytes: implications for integrase inhibitors.

Recently developed integrase inhibitors targeting the HIV-1 integrase (IN) protein block integration of HIV DNA in the target cell, preventing subsequent virus replication. In the absence of integration, viral DNA is shunted towards the formation of extrachromosomal DNA (E-DNA). Although HIV-1 E-DNA does not support productive replication, it is transcriptionally active and produces viral proteins. However, the significance of E-DNA in virus replication and pathogenesis is poorly understood. In this study, the functional activity of the HIV-1 Nef protein expressed in the absence of viral integration was analysed. Using both a recombinant HIV-1 IN defective virus and a diketo acid IN inhibitor, evidence was provided showing that Nef expressed from E-DNA downregulates CD4 surface expression on primary CD4(+) T lymphocytes. These results suggest that proteins expressed in the absence of integration may have potential clinical consequences, an issue that should be further explored with the introduction of IN inhibitors.

CD4 Antigens↗

PubMed2005

Candidate sulfonated and sulfated topical microbicides: comparison of anti-human immunodeficiency virus activities and mechanisms of action.

Poly(styrene 4-sulfonate), cellulose sulfate, polymethylenehydroquinone, and PRO 2000 are sulfated or sulfonated polymers (SPs) under development as topical microbicides. They are presumed to work through similar mechanisms of action, although to date there has been no extensive comparison of their anti-human immunodeficiency virus activities. To determine whether any of these candidate microbicides offers a potential advantage, their in vitro activities, mechanisms of action, stabilities in biological secretions, and toxicities were compared. All four compounds were found to be active against X4, R5, and dualtropic primary isolates and against X4 and R5 laboratory-adapted strains in CD4+ T cells, macrophages, and single-coreceptor cell lines. Our single-cycle experiments using pseudotyped virus suggest that all four SPs function at the binding and entry stages of the viral life cycle but differ in degree of postentry effect. Surface plasmon resonance analyses demonstrate that SPs bind to X4 and R5 monomeric glycoprotein 120 with similar high binding affinities. When mixed with cervicovaginal lavage fluid, SPs maintain inhibitory activity at concentrations achievable in formulations.

Administration, Topical↗

PubMed2005

Dual role of alpha-defensin-1 in anti-HIV-1 innate immunity.

Alpha-defensins are abundant antimicrobial peptides in polymorphonuclear leukocytes and play an important role in innate immunity. We have previously shown that alpha-defensin-1 can inhibit HIV-1 replication following viral entry. Here we examined the molecular mechanism(s) of alpha-defensin-1-mediated HIV-1 inhibition. Alpha-defensin-1 had a direct effect on HIV-1 virions at a low MOI in the absence of serum. The direct effect on HIV-1 virions was abolished by the presence of serum or an increase in virus particles. Studying the kinetics of the HIV life cycle revealed that alpha-defensin-1 inhibited steps following reverse transcription and integration. Analysis of PKC phosphorylation in primary CD4+ T cells in response to alpha-defensin-1 indicated that alpha-defensin-1 inhibited PKC activity. Pretreatment of infected CD4+ T cells with a PKC activator, bryostatin 1, partially reversed alpha-defensin-1-mediated HIV inhibition. Like alpha-defensin-1, the PKC isoform-selective inhibitor Go6976 blocked HIV-1 infection in a dose-dependent manner. Furthermore, kinetic studies and analysis of HIV-1 products indicated that alpha-defensin-1 and Go6976 blocked HIV-1 infection at similar stages in its life cycle, including nuclear import and transcription. Taken together, our studies demonstrate that, in the absence of serum, alpha-defensin-1 may act directly on the virus, but, in the presence of serum, its effects are on the cell, where it inhibits HIV-1 replication. At least 1 of the cellular effects associated with HIV inhibition is interference with PKC signaling in primary CD4+ T cells. Studying the complex function of alpha-defensin-1 in innate immunity against HIV has implications for prevention as well as therapeutics.

Antiviral Agents↗

PubMed2004

Persulfated molecular umbrellas as anti-HIV and anti-HSV agents.

A series of persulfated molecular umbrellas have been synthesized from putrescine, spermidine, spermine, lysine, and cholic acid (1a, 2a, 3a, 4a, and 5a) and their anti-HIV and anti-HSV activities determined. Despite it size, the most active of these conjugates (5a) was able to cross phospholipid bilayers made from 1-palmitoyl-2-oleyol-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG). The unique combination of anti-viral activity, an ability to cross hydrophobic barriers, a lack of cytotoxicity, and a simple three-step synthesis from biogenic starting material suggests that 5a and related conjugates may be exploitable as a novel class of anti-viral agents for systemic and topical applications.

Anti-HIV Agents↗